Conformational dynamics modulate the catalytic activity of the molecular chaperone Hsp90.
Ontology highlight
ABSTRACT:
SUBMITTER: Mader SL
PROVIDER: S-EPMC7075974 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The ubiquitous molecular chaperone Hsp90 makes up 1-2% of cytosolic proteins and is required for viability in eukaryotes. Hsp90 affects the folding and activation of a wide variety of substrate proteins including many involved in signaling and regulatory processes. Some of these substrates are implicated in cancer and other diseases, making Hsp90 an attractive drug target. Structural analyses have shown that Hsp90 is a highly dynamic and flexible molecule that can adopt a wide variety of structurally distinct states. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis only shift the equilibria between a pre-existing set of conformational states. For bacterial, yeast and human Hsp90, there is a conserved three-state (apo-ATP-ADP) conformational cycle; however; the equilibria between states are species specific. In eukaryotes, cytosolic co-chaperones regulate the in vivo dynamic behavior of Hsp90 by shifting conformational equilibria and affecting the kinetics of structural changes and ATP hydrolysis. In this review, we discuss the structural and biochemical studies leading to our current understanding of the conformational dynamics of Hsp90, as well as the roles that nucleotide, co-chaperones, post-translational modification and substrates play. This view of Hsp90's conformational dynamics was enabled by the use of multiple complementary structural methods including, crystallography, small-angle X-ray scattering (SAXS), electron microscopy, Förster resonance energy transfer (FRET) and NMR. Finally, we discuss the effects of Hsp90 inhibitors on conformation and the potential for developing small molecules that inhibit Hsp90 by disrupting the conformational dynamics.
| S-EPMC5070531 | biostudies-literature
Project description:The heat shock protein 90 (Hsp90) is a molecular chaperone central to client protein folding and maturation in eukaryotic cells. During its chaperone cycle, Hsp90 undergoes ATPase-coupled large-scale conformational changes between open and closed states, where the N-terminal and middle domains of the protein form a compact dimerized conformation. However, the molecular principles of the switching motion between the open and closed states remain poorly understood. Here we show by integrating atomistic and coarse-grained molecular simulations with small-angle X-ray scattering experiments and NMR spectroscopy data that Hsp90 exhibits rich conformational dynamics modulated by the charged linker, which connects the N-terminal with the middle domain of the protein. We show that the dissociation of these domains is crucial for the conformational flexibility of the open state, with the separation distance controlled by a β-sheet motif next to the linker region. Taken together, our results suggest that the conformational ensemble of Hsp90 comprises highly extended states, which could be functionally crucial for client processing.
| S-EPMC9251789 | biostudies-literature
Project description:Osmolytes are small molecules that play a central role in cellular homeostasis and the stress response by maintaining protein thermodynamic stability at controlled levels. The underlying physical chemistry that describes how different osmolytes impact folding free energy is well understood, however little is known about their influence on other crucial aspects of protein behavior, such as native-state conformational changes. Here we investigate this issue with the Hsp90 molecular chaperone, a large dimeric protein that populates a complex conformational equilibrium. Using small angle X-ray scattering we observe dramatic osmolyte-dependent structural changes within the native ensemble. The degree to which different osmolytes affect the Hsp90 conformation strongly correlates with thermodynamic metrics of their influence on stability. This observation suggests that the well-established osmolyte principles that govern stability also apply to large-scale conformational changes, a proposition that is corroborated by structure-based fitting of the scattering data, surface area comparisons and m-value analysis. This approach shows how osmolytes affect a highly cooperative open/closed structural transition between two conformations that differ by a domain-domain interaction. Hsp90 adopts an additional ligand-specific conformation in the presence of ATP and we find that osmolytes do not significantly affect this conformational change. Together, these results extend the scope of osmolytes by suggesting that they can maintain protein conformational heterogeneity at controlled levels using similar underlying principles that allow them to maintain protein stability; however the relative impact of osmolytes on different structural states can vary significantly.
| S-EPMC2817839 | biostudies-literature
Project description:Hsp90 is a molecular chaperone essential for protein folding and activation in normal homeostasis and stress response. ATP binding and hydrolysis facilitate Hsp90 conformational changes required for client activation. Hsp90 plays an important role in disease states, particularly in cancer, where chaperoning of the mutated and overexpressed oncoproteins is important for function. Recent studies have illuminated mechanisms related to the chaperone function. However, an atomic resolution view of Hsp90 conformational dynamics, determined by the presence of different binding partners, is critical to define communication pathways between remote residues in different domains intimately affecting the chaperone cycle. Here, we present a computational analysis of signal propagation and long-range communication pathways in Hsp90. We carried out molecular dynamics simulations of the full-length Hsp90 dimer, combined with essential dynamics, correlation analysis, and a signal propagation model. All-atom MD simulations with timescales of 70 ns have been performed for complexes with the natural substrates ATP and ADP and for the unliganded dimer. We elucidate the mechanisms of signal propagation and determine "hot spots" involved in interdomain communication pathways from the nucleotide-binding site to the C-terminal domain interface. A comprehensive computational analysis of the Hsp90 communication pathways and dynamics at atomic resolution has revealed the role of the nucleotide in effecting conformational changes, elucidating the mechanisms of signal propagation. Functionally important residues and secondary structure elements emerge as effective mediators of communication between the nucleotide-binding site and the C-terminal interface. Furthermore, we show that specific interdomain signal propagation pathways may be activated as a function of the ligand. Our results support a "conformational selection model" of the Hsp90 mechanism, whereby the protein may exist in a dynamic equilibrium between different conformational states available on the energy landscape and binding of a specific partner can bias the equilibrium toward functionally relevant complexes.
| S-EPMC2649446 | biostudies-literature
Project description:The molecular chaperones of the Hsp90 family are essential in all eukaryotic cells. They assist late folding steps and maturation of many different proteins, called clients, that are not related in sequence or structure. Hsp90 interaction with its clients appears to be coupled to a series of conformational changes. Using hydrogen exchange mass spectrometry (HX-MS) we investigated the structural dynamics of human Hsp90β (hHsp90) and yeast Hsp82 (yHsp82). We found that eukaryotic Hsp90s are much more flexible than the previously studied Escherichia coli homolog (EcHtpG) and that nucleotides induce much smaller changes. More stable conformations in yHsp82 are obtained in presence of co-chaperones. The tetratricopeptide repeat (TPR) domain protein Cpr6 causes a different amide proton protection pattern in yHsp82 than the previously studied TPR-domain protein Sti1. In the simultaneous presence of Sti1 and Cpr6, protection levels are observed that are intermediate between the Sti1 and the Cpr6 induced changes. Surprisingly, no bimodal distributions of the isotope peaks are detected, suggesting that both co-chaperones affect both protomers of the Hsp90 dimer in a similar way. The cochaperones Sba1 was found previously in the crystal structure bound to the ATP hydrolysis-competent conformation of Hsp90, which did not allow to distinguish the mode of Sba1-mediated inhibition of Hsp90's ATPase activity by stabilizing the pre- or post-hydrolysis step. Our HX-MS experiments now show that Sba1 binding leads to a protection of the ATP binding lid, suggesting that it inhibits Hsp90's ATPase activity by slowing down product release. This hypothesis was verified by a single-turnover ATPase assay. Together, our data suggest that there are much smaller energy barriers between conformational states in eukaryotic Hsp90s than in EcHtpG and that co-chaperones are necessary in addition to nucleotides to stabilize defined conformational states.
| S-EPMC4428384 | biostudies-literature
Project description:BackgroundHsp90 is an essential molecular chaperone that is also a novel anti-cancer drug target. There is growing interest in developing new drugs that modulate Hsp90 activity.Methodology/principal findingsUsing a virtual screening approach, 4-hydroxytamoxifen, the active metabolite of the anti-estrogen drug tamoxifen, was identified as a putative Hsp90 ligand. Surprisingly, while all drugs targeting Hsp90 inhibit the chaperone ATPase activity, it was found experimentally that 4-hydroxytamoxifen and tamoxifen enhance rather than inhibit Hsp90 ATPase.Conclusions/significanceHence, tamoxifen and its metabolite are the first members of a new pharmacological class of Hsp90 activators.
| S-EPMC2848575 | biostudies-literature
Project description:Hsp90 is a ubiquitous molecular chaperone. Previous structural analysis demonstrated that Hsp90 can adopt a large number of structurally distinct conformations; however, the functional role of this flexibility is not understood. Here we investigate the structural consequences of substrate binding with a model system in which Hsp90 interacts with a partially folded protein (?131?), a well-studied fragment of staphylococcal nuclease. SAXS measurements reveal that under apo conditions, Hsp90 partially closes around ?131?, and in the presence of AMPPNP, ?131? binds with increased affinity to Hsp90's fully closed state. FRET measurements show that ?131? accelerates the nucleotide-driven open/closed transition and stimulates ATP hydrolysis by Hsp90. NMR measurements reveal that Hsp90 binds to a specific, highly structured region of ?131?. These results suggest that Hsp90 preferentially binds a locally structured region in a globally unfolded protein, and this binding drives functional changes in the chaperone by lowering a rate-limiting conformational barrier.
| S-EPMC3105473 | biostudies-literature
Project description:Hsp90 is a molecular chaperone involved in the activation of numerous client proteins, including many kinases. The most stringent kinase client is the oncogenic kinase v-Src. To elucidate how Hsp90 chaperones kinases, we reconstituted v-Src kinase chaperoning in vitro and show that its activation is ATP-dependent, with the cochaperone Cdc37 increasing the efficiency. Consistent with in vivo results, we find that Hsp90 does not influence the almost identical c-Src kinase. To explain these findings, we designed Src kinase chimeras that gradually transform c-Src into v-Src and show that their Hsp90 dependence correlates with compactness and folding cooperativity. Molecular dynamics simulations and hydrogen/deuterium exchange of Hsp90-dependent Src kinase variants further reveal increased transitions between inactive and active states and exposure of specific kinase regions. Thus, Hsp90 shifts an ensemble of conformations of v-Src toward high activity states that would otherwise be metastable and poorly populated.
| S-EPMC4485149 | biostudies-literature
Project description:The 90-kDa heat shock protein (HSP90) acts as a specific molecular chaperone in the folding and regulates a wide range of associated proteins such as steroid hormone receptors. It is known that HSP90 possesses two different chaperone sites, both in the N- and C-domains, and that the chaperone activity of HSP90 is blocked by binding of geldanamycin (GA) to the N-domain, the same as the ATP-binding site. Here we show that Cisplatin [cis-diamminedichloroplatinum (II), CDDP], an antineoplastic agent, associates with HSP90 and reduces its chaperone activity. In order to analyse the binding proteins, bovine brain cytosols were applied to a CDDP-affinity column and binding proteins were eluted by CDDP. In the elutants, only 90-kDa protein bands were detected on SDS/PAGE, and the protein was cross-reacted with the anti-HSP90 antibody on immunoblotting. No protein bands were detected in the elutants from the control column on SDS/PAGE. These results indicated that CDDP has a high affinity for HSP90. On CD spectrum analysis, the binding of CDDP to HSP90 resulted in a conformational change in the protein. Although HSP90 inhibited the aggregation of citrate synthase as a molecular chaperone in vitro, the activity was suppressed almost completely in the presence of CDDP. Mg/ATP has an influence on the chaperone activity to some extent. The CDDP binding region of HSP90 is near the C-terminal which is quite different from the GA-binding site. Our results suggest that the chaperone activity of HSP90 may be inhibited by the binding of CDDP or GA by different mechanisms.
| S-EPMC1220604 | biostudies-other
Project description:The highly conserved 90 kDa heat shock protein (Hsp90) chaperones use ATP to regulate the stability and activity of many signalling molecules like protein kinases and transcription factors. Studies using crystallography, electron microscopy and small-angle X-ray scattering yielded controversial results for the conformational states that these dimeric multidomain proteins assume while progressing through the ATPase cycle. To better understand the molecular mechanism of Hsp90 proteins, we studied the conformational dynamics of the Escherichia coli homologue HtpG in solution using amide hydrogen exchange mass spectrometry (HX-MS) and fluorescence spectroscopy. A conformation-sensitive fluorescent probe allowed to elucidate the ATPase cycle of HtpG. Continuous-labelling and pulse-labelling HX-MS experiments revealed major ATP-induced conformational changes throughout the protein that do not occur simultaneously, but progress surprisingly slow from the immediate nucleotide-binding site towards the N terminus and the middle domain. The conversion between the different conformational states is rate limiting for ATP hydrolysis, and the nucleotide-coordinating residue, Glu34, is important for the rate constant of conversion. Our findings, for the first time, allow to kinetically resolve changes in the conformational dynamics of individual structural elements of Hsp90.
| S-EPMC2657576 | biostudies-literature